15, and 0.2 μg mL−1.

ROS accumulation in fungal cells was examined using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Torin 1 molecular weight Molecular Probes) (Liu et al., 2010). Conidia of A. niger were cultured in Sabouraud medium 16 h and treated with CTBT (10 μg mL−1) for 3 h at 28 °C. The hyphae were washed and resuspended in 10 mM (PBS) and incubated in 40 mM H2DCFDA for 30 min at 28 °C. Then, the hyphae were washed, resuspended in 10 mM PBS, and visualized by fluorescence microscopy using excitation and emission wavelengths of 480 and 530 nm, respectively. The antifungal activity of CTBT was assessed using the agar diffusion method on Mueller–Hinton medium, as recommended by Espinel-Ingroff et al. (2007). CTBT (10 μg per disk) was found to inhibit the growth of different molds involving both saprophytic and pathogenic fungal species. It induced inhibition zones, varying in diameter from 19 mm for M. gypseum to 50 mm for P. purpurogenum, that were apparently larger than those caused by itraconazole (30 μg per disk) (Table 1). This was probably due to PI3K inhibitor CTBT’s different rate of diffusion into the agar medium. Under the same experimental conditions, fluconazole (25 μg per disk), having only limited activity against filamentous fungi (Loeffler & Stevens, 2003),

did not produce zones of growth inhibition. In further experiments, we used two fungal species, A. niger and A. fumigatus. They represent industrially and medically important molds. Aspergillus niger is used for the production of organic acids and enzymes. Aspergillus fumigatus is a human pathogen that causes invasive, often fatal, pulmonary disease in immunocompromised Prostatic acid phosphatase individuals

(Maschmeyer et al., 2007). As shown in Fig. 1, CTBT added at a concentration of 80 μg mL−1 to Sabouraud broth containing conidia (106 per mL) of A. niger or A. fumigatus, inhibited the swelling of conidia, and prevented germ tube development. After 24 h of interaction of A. niger or A. fumigatus conidia with CTBT (80 μg mL−1), no fungal growth was detected on Sabouraud agar in spots (1.5 × 104 conidia) of treated conidia (Fig. 2), indicating that the effect of CTBT was fungicidal. CTBT has been found to induce superoxide formation and oxidative stress in yeast cells (Batova et al., 2010). Apparently, the same occurs in filamentous fungi. CTBT added to A. niger mycelium induced the ROS formation as detected by H2DCFDA, which is a cell-permeable indicator for ROS. Intense green fluorescence was distributed along the plasma membrane and within the cytoplasm (Fig. 3). However, no ROS-specific signals were observed in control hyphae (Fig. 3). When A. niger or A. fumigatus conidia were applied (in 5 μL) to solid growth media, radial growth of colonies was observed. When using initial spore amounts 2 × 102–2 × 104 conidia, colonies appeared with a diameter of 50 mm after 3–7 days, depending on fungal species and culture media used.